1. Field of the Invention
This invention relates to a modified phytochrome A (PHYA) nucleic acid molecule of which Pr absorption spectra have been shifted to longer wavelength (i.e. bathochromic or red-shifted phytochrome A), in order to give plants shade-tolerance that increases crop yields. The phytochrome A functions as the photoreceptor in far-red wavelength light in mediating the suppression of shade avoidance and the development of plants. These bathochromic phytochromes absorb and utilize light even in the canopy and shade conditions, which suppresses shade avoidance reactions in plants (i.e. shade tolerance). Since the shade avoidance reactions re-distributes energy and resources to unnecessary elongation and acceleration of senescence (i.e. early flowering), it causes drastic reduction in products such as leaves, storage organs and seeds. Thus, the developed bathochromic phytochromes enable us to develop shade tolerant plants with high yields. The present invention also provides the methods and processes for generating transgenic plants transformed with the said nucleic acid molecules to engineer shade tolerance of economically important crop plants for high-yielding.
2. Description of Prior Art
Light is the most important environmental factor for optimal growth and development of plants (Chen et al., 2004). They harness not only energy from light for anabolic pathways that construct their building blocks, but also adapt to changes in light during their life cycle from germination to flowering. Phytochrome is a photoreceptor that manage a variety of photomorphogenic responses to the red/far-red region of the spectrum (Smith, 2000). They are dimeric chromopeptides (monomer sizes of 120˜130 kDa) that carry the chromophore phytochromobilin (PΦB), which is covalently linked to a cysteine residue on each peptide via a thioether linkage. There are two spectrally distinct forms of phytochromes, a red-light (R, λmax=660 nm) absorbing Pr form and a far-red light (FR, λmax=730 nm) absorbing Pfr form (FIG. 1A). The latter form is considered as the active form of phytochrome because of the promotive effect of red-light on most physiological responses. Phytochrome signaling in plants is driven by phototransformation between the two forms (Kim et al., 2002).
Competition for sunlight is one of the most important aspects in regulating plant development (Ballare, 1999). Plants grown under dense canopies or at high density (i.e. shaded conditions) perceive a decrease in the ratio of R to FR light (R:FR ratio). This change in light quality serves as a warning of competition, triggering a series of responses known collectively as the shade avoidance syndrome or shade avoidance reactions (Smith and Whitelam, 1997; Devlin et al., 2003). The reactions in plants induce a rapid and dramatic increase in the extension growth of stems and petioles at the expense of leaf growth, storage organ production, and reproductive development (Table 1). Prolonged shade causes dramatically accelerated flowering, reduced seed sets, and immature fruits. The shade avoidance reactions are mediated predominantly by phytochromes (Smith and Whitelam, 1997). Phytochromes respond to the R:FR ratio as an indicator of proximity to and shade from neighbors. Since blue and red lights are selectively absorbed by chlorophylls for photosynthesis, far-red light is relatively enriched in shaded conditions (FIG. 1B). Thus, shade is represented as a low R:FR ratio. The enriched far-red light signal is recognized by phytochromes as a change in photoequilibrium between Pr and Pfr. The photoequilibrium of phytochrome is represented by the ratio [Pfr]/[Ptot], where [Ptot]=[Pr]+[Pfr]. The lowered R:FR ratio induces a decrease in the Pfr form of phytochromes, which trigger the shade avoidance reactions in plants.
TABLE 1Shade avoidance reactions in plants.Physiological processResponse to shadeGerminationRetardationExtension growthAccelerationLeaf developmentRetardationChloroplast developmentRetardationBranchingRetardationFloweringAccelerationStorage organ depositionSevere reduction
In monocultures of crop plants in close proximity, competition for light is an important factor in determining crop yields, because it induces shade avoidance reactions such as elongation of internodes and petioles, inhibition of leaf expansion and growth, retardation of chloroplast development, and early flowering. Since the shade avoidance reaction re-distributes energy and resources to unnecessary elongation and acceleration of senescence (FIG. 2), it cause drastic reduction in products such as leaves, storage organs and seeds. The overexpression of phytochromes in crop plants has been used to overcome these losses from shade avoidance reactions (Robson et al., 1996; Robson and Smith, 1997). FR light is enriched in the shade (See FIG. 1), so the R:FR ratio is decreased. Phytochrome B (phyB) perceives the low R:FR ratio and rapidly induces shade avoidance reactions (Robson et al., 1993). Phytochrome A (phyA) has an antagonistic function to phyB in shade avoidance reactions (Botto et al., 1996). Thus, the Avena (oat) phyA gene has been introduced into crop plants such as tobacco, tomato, potato and wheat (Boylan and Quail, 1989; Heyer et al., 1995; Robson et al., 1996; Sineshchekov et al., 2001; Shlumukov et al., 2001). When constitutively expressed, the Avena phyA increases shade tolerance, resulting in improvements of leaf expansion and growth without the expense of elongation growth. Consequently, Avena phyA-overexpressing tobaccos showed shortened stature in low R:FR and proximity-conditional dwarfism in dense culture. The harvest index of transgenic tobacco showed approximately 20% improvement in leaf product (Robson et al., 1996). Also, transgenic tomatoes, potatoes and wheat displayed suppression of shade avoidance with improved leaf expansion and growth, greening and increased harvest indices of storage organs or seeds (Boylan et al., 1991; Heyer et al., 1995; Shlumukov et al., 2001).
The increase of shade tolerance by overexpression of phytochromes is limited because of the limitation of expression levels and also the degradation of phytochrome proteins upon light illumination. Theoretically, an increase in Pfr under low R:FR ratios could mediate shade tolerance in plants. Thus, spectral phytochrome mutants that absorb longer wavelengths (i.e. bathochromic or red-shifted mutants in Pr absorption maxima) can be used to confer shade tolerance on plants. As shown in FIG. 3, the simulated spectra of Pr in bathochromic mutants shows an increase in the area overlapping with the shade spectrum, in which phytochromes recognize shade like red light and can be transformed to more Pfr form. Consequently, a shift of photoequilibrium to Pfr would overcome the shade avoidance. The absorption spectra of the phytochromes overlapped with the shade spectrum and the red-shifted mutants had greater overlap than the wild-type or blue-shifted mutant (FIG. 3). The overlap of the 8 nm and 12 nm red-shifted mutants increased to 153±4% and 176±4% respectively, whereas the 8 nm blue-shifted mutant decreased to 67±4%. These calculations suggest that the red-shifted mutant can absorb more light in the shade, and maintain an increased amount of biologically active Pfr compared to wild-type phyA, thus conferring shade tolerance to plants. Thus, bathochromic phytochromes in this invention can be practically applied to suppress shade avoidance reactions under shaded conditions such as canopy and proximity cultures, which increase shade tolerance to plants with high yields. The plants referred to here are those economically important in agriculture and horticulture. As used herein, the term “economically important higher plants” refers to higher plants that are capable of photosynthesis and widely cultivated for commercial purpose. The term “plant cell” includes any cells derived from a higher plant, including differentiated as well as undifferentiated tissues, such as callus and plant seeds.